Proc. Natl. Acad. Sci. USAVol. 93, pp. 11913-11918, October 1996Microbiology

Haemophilus influenzae pili are composite structures assembledvia the HifB chaperone

(organelle biogenesis/pathogenesis)

JOSEPH W. ST. GEME III*tt, JEROME S. PINKNERt, GRAHAM P. KRASAN*, JOHN HEUSER§, ESTHER BULLITTr,ARNOLD L. SMITHII, AND Scorr J. HULTGRENt*Edward Mallinckrodt Department of Pediatrics, and Departments of tMolecular Microbiology and §Cell Biology and Physiology, Washington University Schoolof Medicine, St. Louis, MO 63110; 1Department of Biophysics, Boston University School of Medicine, Boston, MA 02118; and IlDepartment of MolecularMicrobiology and Immunology, University of Missouri, Columbia, MO 65203

Communicated by Stanley Falkow, Stanford University School of Medicine, Stanford, CA, August 5, 1996 (received for review April 19, 1996)

ABSTRACT Haemophilus influenzae is a Gram-negativebacterium that represents a common cause of human disease.Disease due to this organism begins with colonization of theupper respiratory mucosa, a process facilitated by adhesivefibers called pili. In the present study, we investigated thestructure and assembly of H. influenzae pili. Examination ofpili by electron microscopy using quick-freeze, deep-etch andimmunogold techniques revealed the presence of two distinctsubassemblies, including a flexible two-stranded helical rodcomprised of HifA and a short, thin, distal tip structurecontaining HiMD. Genetic and biochemical studies demon-strated that the biogenesis ofH. influenzae pili is dependent ona periplasmic chaperone called HifB, which belongs to thePapD family of immunoglobulin-like chaperones. HifB bounddirectly to HifA and HifD, forming HifB-HifA and HifB-HifDcomplexes, which were purified from periplasmic extracts byion-exchange chromatography. Continued investigation of thebiogenesis ofH. influenzae pili should provide general insightsinto organelle development and may suggest novel strategiesfor disease prevention.

Mucosal colonization is an essential early step in the patho-genesis of most bacterial diseases (1). Bacterial attachment tohost epithelial cells is a key event in this process and ismediated by specialized proteins called adhesins, which areusually components of hair-like surface fibers (2).Among Gram-negative bacteria, adhesive fibers are most

often assembled in a process that involves an immunoglobulin-like periplasmic chaperone and an outer membrane proteincalled an usher (2). Fibers assembled by the chaperone/usherpathway have diverse molecular architectures ranging fromrod-like cylinders called pili to very thin flexible filaments thatcoil into an amorphous mass on the bacterial surface (3). Atleast some adhesive fibers are composite structures consistingof rod-like subassemblies joined distally to very thin fibrillae(tip fibrillae) (4, 5).The prototype periplasmic chaperone is PapD, which is

required for assembly of P pili in uropathogenic Escherichiacoli. This protein has been shown to have an immunoglobulin-like three-dimensional structure and functions by preventingpremature subunit assembly in the periplasm (6, 7). PapDbinds to and caps interactive surfaces on subunits by formingchaperone-subunit complexes, which are then escorted toouter membrane assembly sites comprised of the PapC usher(7, 8). Subsequently, complexes are dissociated, and subunits areincorporated into pili across the outer membrane. In the absenceof an interaction with PapD, pilus subunits misfold and formoff-pathway aggregates that are proteolytically degraded (7).

Haemophilus influenzae is a Gram-negative coccobacillusthat represents a frequent etiology of both localized respira-tory tract and systemic disease (9). Disease due to H. influenzaebegins with colonization of the nasopharynx, followed byeither contiguous spread within the respiratory tract or inva-sion of the bloodstream. In 1982 two groups reported thepresence of pili on selected H. influenzae isolates (10, 11).These structures were found to promote attachment to humanoropharyngeal epithelial cells and to mediate agglutination ofhuman erythrocytes expressing the AnWj antigen (12). Severalstudies have demonstrated that pili enhance adherence tohuman nasopharyngeal and nasal turbinate tissue in organculture (13-15). More recently, Weber et al. (16) observed thatpili play an important role in colonization using the monkeynasopharyngeal colonization model.

Genetic evidence suggests that the biogenesis of H. influ-enzae pili depends on the chaperone/usher pathway. The genecluster required for assembly of pili has been cloned andincludes a gene designated hifA, which encodes the majorstructural subunit (HifA) of the pilus (17). The remainder ofthe cluster is located upstream of hifA and is transcribeddivergently, beginning with a chaperone-like gene designatedhifB, followed by an usher-like gene, hifC, and then two geneswith unknown functions, hifD and hifE (17-20). A detailedanalysis of the architecture of H. influenzae pili has beenlacking. Similarly, little information exists regarding the pro-tein-protein interactions involved in the biogenesis of theseappendages. The present study was undertaken to investigatethe structure and assembly of H. influenzae pili.

MATERIALS AND METHODSBacterial Strains and Plasmids. H. influenzae strain Eagan

is a type b strain that was originally isolated from the cere-brospinal fluid of a child with meningitis. The nucleotidesequences of the hifA-E genes in this strain have been reported(refs. 19-21; A.L.S., unpublished work). E. coli strains DH5a,ORN103, and C600 have been previously described (22, 23).

Plasmid pMCC10 contains the hifA and hifB genes and the5' portion of the hifC gene from strain Eagan. PlasmidpABK15 is a derivative of pMCC10 with the kanamycincassette from pUC4K (24) inserted at theXhoI site within hifB.Plasmid pDC101 is also a derivative of pMCC10 and containsa kanamycin cassette inserted at the EcoRI site within hifC.Plasmid pJS201 contains hifA by itself downstream of the trcpromoter in pTrc99A. The hifA gene was amplified frompABK15 using the polymerase chain reaction (PCR) andprimers that correspond to nucleotides 73-92 bases upstream

Abbreviations: IPTG, isopropyl ,3-D-thiogalactoside; FPLC, fast pro-tein liquid chromatography.*To whom reprint requests should be addressed at: Department ofMolecular Microbiology, Washington University School of Medicine,660 South Euclid Avenue, Box 8230, St. Louis, MO 63110. e-mail:stgeme@borcim.wustl.edu.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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11914 Microbiology: St. Geme et al.

of the hifA start codon and 34-54 bases downstream of the stopcodon. Plasmid pJS202 contains hifB by itself downstream ofthe tac promoter in pMMB91. The hifB gene was amplifiedfrom strain Eagan chromosomal DNA using the PCR andprimers corresponding to sequences 53-72 nt upstream of thestart codon and 27-46 nt downstream of the stop codon.Plasmid pGK101 contains hifD by itself downstream of the trcpromoter in pTrc99A. The hifD gene was amplified fromEagan chromosomal DNA using the PCR and primers thatcorrespond to the 5' end and 3 bases upstream and the 3' endand 3 bases downstream of hifD.

Purification of H. influenzae Pili. To purify pili from H.influenzae strain Eagan, bacteria were incubated in supplementedbrain heart infusion broth (25) at 37°C overnight. Cells were thencentrifuged at 8000 x g at 4°C for 20 min and were resuspendedin 0.1 M Tris buffer (pH 8). Pili were sheared from the cell surfaceby blending at low speed for 10 min in an Omnimixer (OmniInternational, Waterbury, CT) immersed in ice. Following cen-trifugation at 25,000 x g at 4°C for 60 min, the supernatant wasrecovered and precipitated with 10% saturated ammonium sul-fate. The resulting solution was centrifuged at 15,000 x g at 4°Cfor 20 min, and the supernatant was then precipitated with 45%saturated ammonium sulfate. Precipitation was repeated with10% and then 45% saturated ammonium sulfate, and the finalpilus pellet was resuspended in 0.5 M Tris (pH 7).

Purification of HifB, HifB-HifA Complex, and Hifm-HifDComplex. HifB was purified from E. coli C600 carrying pJS202(hifB). Bacteria were grown in 20 liters of Luria-Bertani brothto an optical density at 600 nm of 1.0 and were then inducedwith 0.25 mM isopropyl ,B-D-thiogalactoside (IPTG) for 1 h.Subsequently periplasmic proteins were extracted, concen-trated with 75% saturated ammonium sulfate, and dialyzedagainst 20 mM Mes (pH 6.5). The resulting material wasfractionated on a fast protein liquid chromatography (FPLC)HiLoad S Sepharose cation exchange column and eluted witha gradient of 0 to 1 M KCl. The fractions containing HifB werepooled and dialyzed against 50 mM phosphate, pH 7.0/1 Mammonium sulfate and then fractionated on an FPLC phenylsuperose hydrophobic interaction column.HifB-HifA complex was purified from strain C600 carrying

pJS201 (hifA) and pJS202 (hifB). Bacteria were grown andinduced as described in the previous paragraph. Followinginduction, periplasmic proteins were extracted, concentratedwith 75% saturated ammonium sulfate, dialyzed against 20mM Tris (pH 7.5), and then collected as flow-through on anFPLC HiLoad Q Sepharose anion exchange colum. Thefractions containing HifB-HifA complex were pooled anddialyzed against 20 mM Mes (pH 6.5). The resulting samplewas then fractionated on an FPLC HiLoad S Sepharose cationexchange column, eluting with a gradient of 0 to 1 M KCl.HifB-HifD complex was purified from strain C600 harbor-

ing pGK101 (hifD) and pJS202 (hifB) using the protocoldescribed for HifB-HifA complex.

Electron Microscopy. Freeze etch. Purified pili were pre-pared for electron microscopy by adsorption to mica chipswhich were quick-frozen, then freeze-fractured and deep-etched, and finally rotary replicated with platinum (26, 27).Whole bacteria were prepared for examination by incubatingin supplemented brain heart infusion broth to log phase andthen washing once with phosphate-buffered saline (PBS).Subsequently bacteria were adsorbed to glass coverslips whichwere quick-frozen, completely freeze-dried, and then rotaryreplicated with platinum (27).

Negative staining. Bacteria were grown on chocolate agarplates for "20 h, then suspended in PBS, and negativelystained with 0.5% uranyl acetate as described (25). Proteinpreparations were diluted to a concentration of "10 ,ug/ml,then spotted onto carbon-coated 300 mesh copper grids,washed with eight drops of buffer, and stained with 2% uranylacetate for 15 sec.

Immunolabeling. Bacteria were prepared for immunoelec-tron microscopy as described by Roberts et al. (28) with minormodifications. In brief, glow-discharged carbon-coated, form-var-strengthened grids were overturned on a drop of bacterialsuspension and incubated for 2 min. Subsequently, sampleswere blocked for 1 h with PBS containing 23% goat serum and0.1% gelatin and were then incubated for 2 h with a 1:25dilution of primary antibody. Next, samples were washed withPBS and incubated for 1 h with a 1:25 dilution of goatanti-rabbit IgG conjugated to 12-nm colloidal gold beads(Jackson ImmunoResearch). Samples were then washed againwith PBS, fixed with 1% glutaraldehyde in PBS, and negativelystained with 0.5% uranyl acetate.

Cell Fractionation and Protein Analysis. Whole-cell lysateswere prepared by resuspending bacterial pellets in water andan equal volume of 2x Laemmli buffer (29). Periplasmicproteins were extracted from H. influenzae using polymyxin Bsulfate, as described (30). Periplasmic proteins were recoveredfrom E. coli derivatives according to the method of Slonim etal. (31). Proteins were resolved by SDS/PAGE using 10%-12.5% acrylamide gels (29). Western blots were performedwith mouse ascites fluid AF100 raised against denatured HifAor rabbit antiserum raised against purified HifB, HifB-HifAcomplex, denatured HiMD, or HifB-HifD complex. DenaturedHifA was obtained by resolving purified pili on an SDS/polyacrylamide gel and excising the monomeric HifA band,while denatured HifD was prepared by resolving purifiedHifB-HifD complex by SDS/PAGE and excising the HifDband. The excised material was minced and then used toimmunize animals.Recombinant DNA Methods. DNA ligations, restriction

endonuclease digestions, and gel electrophoresis were per-formed according to standard techniques (22). In H. influen-zae, transformation was performed using the MIV method ofHerriott et al. (32).

Construction ofHiB- and HifC- Mutants. Mutagenesis ofhifB involved transformation of strain Eagan with linearizedpABK15, followed by selection with kanamycin. Similarly,insertional inactivation of hifC was accomplished by trans-forming strain Eagan with linearized pDC101 and selectingwith kanamycin. Allelic exchange was confirmed by Southernhybridization, probing with the kanamycin cassette frompUC4K (24) and hifB or hifC, as appropriate.

RESULTSH. influenzae Pili Are Composite Structures. To assess the

structural features of H. influenzae pili, purified pili from strainEagan were examined by quick-freeze, deep-etch electronmicroscopy (Fig. 1). As shown in row 1 of Fig. 1, inspection athigh magnification revealed a relatively thick rod and a shortthin tip differentiation reminiscent of the tip fibrillum seen onP and type 1 pili (4, 5). Consistent with observations made byconventional electron microscopy, pilus rods displayed manybends and flexures, implying that they are flexible. A numberof pilus tips were noted to form curved hooks, suggesting thatthese structures are flexible as well.

Close examination of replicas of pilus rods revealed adiameter of 8-10 nm; subtraction of platinum shadowingallowed the calculation of a true diameter of 6-7 nm. Inaddition, these structures were noted to have regularly spacedhorizontal striations indicative of a tight helical architecture.Interestingly, a cross-over repeat, consistent with a two-stranded helical structure was observed. This cross-over repeatwas best appreciated in fields with multiple fibers alignedalongside one another (Fig. 1, row 2). The single-start helix wasdetermined to be left-handed with a repeat of 5.2 nm, while thedouble-start helix was found to be right-handed with a cross-over repeat of 26 nm. Using these repeat dimensions, pilus rodswere calculated to have five subunits per double-start half-turn. Overall, H. influenzae pilus rods showed a striking

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Proc. Natl. Acad. Sci. USA 93 (1996) 11915

FIG. 1. Freeze-etch electron microscopy of purified pili and fila-mentous actin. Row 1 depicts the ends of purified pili from H.influenzae strain Eagan, highlighting very short tip differentiations.The closely spaced horizontal striations in the pilus rod are charac-teristic of a tight helix. Row 2 shows alignment of pili either two abreast(panels 1-4), four abreast (panel 5), or five abreast (panel 6); thebleed-over of the platinum coating between adjacent pili highlights thepresence of a cross-over repeat, which is indicative of a two-strandedhelical architecture. Row 3 depicts F-actin from rabbit skeletal musclefor comparison; note the striated appearance and cross-over repeat.Again the bleed-over of the platinum coating between actin filamentshighlights the cross-over repeat. (x 192,000.)

resemblance to filamentous actin (Fig. 1, row 3), which isknown to be a two-stranded structure with actin monomersarranged in a single-start left-handed helix and a double-startright-handed helix.

Analysis of pilus tips revealed a diameter of -2.5 nm and anaverage length of roughly 16 nm. Based on examination ofrelatively straight tips, there was little variation in length fromone tip to another.Whole bacteria were also examined using the quick-freeze,

deep-etch technique. The structures emanating from the sur-face of whole organisms had the same morphologic features aspurified pili, including a striated appearance and a 26-nmcross-over repeat (not shown). Since a relatively low resolutionprotocol was required for these studies, it was difficult toresolve the presence of tip differentiations.HifB Is a Periplasmic Chaperone That Stabilizes HiA.

Given the apparent two-stranded architecture of H. influenzaepili, we wondered whether the general principles that governthe chaperone/usher assembly pathway would still apply. TheHaemophilus pilus gene cluster includes a gene designated hifBwhich encodes a product (HifB) with homology to PapD (17).Molecular modeling has suggested that HifB is a member ofthe PapD family of immunoglobulin-like chaperones (6).

Using E. coli C600 harboring pJS202 (hiJB), HifB was overex-pressed and then purified from periplasmic extracts (see Fig. 3A,lane 1, and Fig. 6A, lane 6). N-terminal amino acid sequencing of

the purified protein revealed the sequence -VIITGTGT, whichcorresponds to residues beginning at position 25 in the predictedHifB polypeptide. (The identity of the first residue could not bedetermined because of excessive background.) The cleaved 24-amino acid fragment has features of a typical prokaryotic signalpeptide and terminates with the sequence ANA. Examination ofpurified HifB by isoelectric focusing demonstrated a pl of >9 (notshown), similar to that of PapD and other chaperones belongingto the PapD family. Cell fractionation studies performed withanti-HifB antiserum confirmed that in H. influenzae strain Eaganand in E. coli C600/pJS202, HifB was most abundant in theperiplasm and was absent from the outer membrane (not shown).

Insertional inactivation of the hifB gene in H. influenzaestrain Eagan eliminated expression of pili on the surface of theorganism and abolished bacterial-mediated hemagglutination(not shown). As shown in Fig. 2A, examination of this strain byimmunoblotting with anti-HifA antibody revealed no detect-able HifA in either periplasmic extracts or whole cell lysates.In contrast, insertional inactivation of hifC, the downstreamgene in the hif gene cluster, had no effect on the quantity ofHifA in the periplasm or in whole cells (Fig. 2A). These resultssuggested that HifB serves to stabilize HifA against degrada-tion. To pursue this consideration further, clones containingeither hifA (pJS201) or hifB (pJS202) or both were introducedinto E. coli ORN103. Following induction of gene expressionwith IPTG, periplasmic fractions were isolated from each ofthese strains and examined for reactivity with antibody againstHifA. As shown in Fig. 2B, HifA could not be detected in E.coli harboring hifA alone but was easily visualized in E. colicarrying both hifA and hifB. E. coli harboring hifB alone failedto react with antibody against HifA.HifB-HifA Complexes Are the Major Building Blocks of

Pilus Rods. The observation that HifB functions to stabilizeHifA suggested that these two proteins interact with one another

A 1 2 3 4 5 6 7 8

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FIG. 2. Western analysis of H. influenzae strain Eagan and isogenichifB and hifC mutants and of E. coli ORN103 carrying pJS201 (hifA),pJS202 (hifB), or both, blotting with anti-HifA antibody. (A) Whole-cell lysates and periplasms from strain Eagan derivatives. Lanes: 1,whole-cell lysate from piliated wild-type strain; 2, whole-cell lysatefrom nonpiliated phase variant; 3, whole-cell lysate from HifB mutant;4, whole-cell lysate from HifC mutant; 5, periplasm from piliatedwild-type strain; 6, periplasm from nonpiliated phase variant; 7,periplasm from HifB mutant; 8, periplasm from HifC mutant. (B)Periplasms from E. coli ORN103 derivatives. Lanes: 1, E. coliORN103/pJS201; 2, E. coli ORN103/pJS202; 3, E. coli ORN103/pJS201 plus pJS202; 4, purified pili.

Microbiology: St. Geme et al.

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11916 Microbiology: St. Geme et al.

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FIG. 3. Purification of HifB-HifA complex from periplasm of E.coli C600 carrying pJS201 (hifA) and pJS202 (hifB). Lanes: 1, purifiedHifB; 2, material purified from E. coli C600 (pJS201 plus pJS202). (A)Native polyacrylamide gel stained with Coomassie blue. (B) Nativepolyacrylamide gel examined by Western blot with antibody againstHifB. (C) Native polyacrylamide gel examined by Western blot withantibody against HifA.

directly. To investigate this possibility, we attempted to demon-strate a HifB-HifA complex in the periplasmic fraction from E.coli C600 carrying pJS201 (hifA) and pJS202 (hi/B). Periplasmicproteins from this strain were subjected to anion exchangechromatography, and fractions that contained both HifB andHifA were subsequently subjected to cation exchange chroma-tography. Analysis of the resulting material on a native polyacryl-amide gel followed by staining with Coomassie blue revealed onepredominant protein band and three minor bands (Fig. 3). Thepredominant band reacted with both anti-HifB and anti-HifAantibodies, indicating that this band represents a HifB-HifAcomplex. Of the three minor bands, one migrated to the sameposition as purified HifB and reacted only with anti-HifB anti-serum, arguing that this band represents free HifB. The other twominor bands failed to react with either antibody preparation andare presumably contaminating proteins. Analysis of periplasmicextracts from strain Eagan by an analogous approach also dem-onstrated evidence of a HifB-HifA complex, although in smallerquantities (not shown).

Previous studies have suggested that HifA is the major struc-tural subunit of the mature pilus. However, efforts to confirm thisconclusion microscopically have been complicated by the fact thatantiserum raised against HifA excised from a denaturing poly-

A

acrylamide gel fails to react with native pili (33). To circumventthis problem, we generated rabbit antiserum against purifiedHifB-HifA complex. Based on observations with PapD and Ppilus subunits, we assumed that HifB holds HifA in a nativeconformation. The resulting antiserum was adsorbed with Eagan-hif- to remove antibodies against contaminating proteins andwas then used to examine piliated Eagan by immunoelectronmicroscopy. As shown in Fig. 4A, this antiserum decorated theentire shaft of the pilus. In contrast, anti-HifB antiserum failed toreact with pili (Fig. 4B), arguing that the reactivity observed withthe antiserum against HifB-HifA complex was due to antibodiesagainst HifA. In control samples with EaganhiffB-, there was nolabeling with either the anti-HifB-HifA or the anti-HifB anti-serum (not shown).

Chaperones are thought to control pilus assembly by cappingand uncapping the associative surfaces of pilus subunits (7). Inrecent work, the major subassemblies of E. coli P pili, namelythe pilus rod comprised of PapA and the tip fibrillum com-prised of PapE, were reconstituted in vitro from purifiedchaperone-subunit complexes (42). Freeze-thaw techniqueswere used to achieve chaperone uncapping of PapA and PapE,resulting in the formation of pilus rods and tip fibrillae,respectively. To extend these observations, purified HifB-HifA complex was spotted onto carbon-coated grids, stainedwith uranyl acetate, and then examined by transmission elec-tron microscopy. As shown in Fig. 5, numerous short rods weredetected; these rods ranged in length from 10 to 100 nm andwere virtually identical in appearance to mature H. influenzaepilus rods. The short rods presumably represent HifA multimersthat formed after spontaneous dissociation of HifB from HifA.HifB-HifD Complexes Are Precursors of Pilus Tips. The

hifD gene is located immediately downstream ofhifC and encodesa protein with homology to HifA (17,20). Based on this homologyand the presence of a chaperone binding motif at the carboxylterminus, HifD is presumed to be a minor structural subunit (17,20). To investigate this possibility, we began by constructing an E.coli C600 derivative harboring both hifB and hi4D (pJS202 andpGK101). Following induction of gene expression with IPTG,periplasmic proteins were extracted and examined by SDS/PAGE. As shown in Fig. 64, induced proteins corresponding insize to HifB (-25 kDa) and HifD (21-22 kDa) were observed.

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FIG. 4. Immunoelectron micrographs of H. influenzae strain Eagan. Samples were prepared with piliated strain Eagan and antisera againstHifB-HifA complex (A), HifB (B), and HifB-HifD complex (C). (Bar = 110 nm.)

Proc. Natl. Acad. Sci. USA 93 (1996)

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Proc. Natl. Acad. Sci. USA 93 (1996) 11917

FIG. 5. Negatively stained electron micrographs of purified pili versus purified HifB-HifA. (A) Purified pili from H. influenzae strain Eagan.(B) Purified HifB-HifA; note that the short filaments are virtually identical in morphology to pure pili. (Bar = 50 nm.)

Using a scheme similar to that employed to purify HifB-HifAcomplex, we purified HifB-HifD complex by ion exchange chro-matography (Fig. 6A). N-terminal amino acid determinationrevealed that mature HifD begins with the sequence VDGRVT-FQGE, indicating cleavage of a 54-amino acid signal peptide thatterminates with AYA.

In further experiments, HifB-HifD complex was loadedonto a preparative SDS/polyacrylamide gel, and the HifDband was excised and injected into rabbits. As shown in Fig. 6B,the resulting antiserum recognized HifD and also reacted withpurified pili. In contrast, there was no reactivity with HifA(lane 6). Considered together, these findings indicate thatHifD is incorporated into the mature pilus.To localize HifD within the pilus structure, we generated

antiserum against purified HifB-HifD complex and examinedpiliated Eagan by immunoelectron microscopy. As shown inFig. 4C, gold beads were present predominantly at the ends ofpili, suggesting that HifD is a component of the tip differen-tiation at the distal end of the pilus. In a control sample withEaganhif-, we observed no labeling (not shown).

DISCUSSIONQuick-freeze deep-etch electron microscopy has been em-ployed previously to examine P and S pili (4, 34), which areexpressed exclusively by E. coli, and type 1 pili (5), which arecommon to members of the family Enterobacteriaceae. Thesefibers are composite structures consisting of a single-strandedhelical rod that emanates from the bacterial surface andconnects to a distally located tip fibrillum. P pilus and type 1pilus rods have been characterized in detail, and their helicalsymmetry is known to be right-handed (35, 36). H. influenzaepili represent the first pilus organelles outside the familyEnterobacteriaceae to be examined by high resolution electronmicroscopy. Our results indicate that H. influenzae pili arecomposite structures as well. However, their architecture isdistinctive, with the pilus rod consisting of a single-startleft-handed helix and a double-start right-handed helix.

In general, the molecular architecture of a pilus is determinedby the structural properties and interactive surfaces of the sub-units (42). Bullitt and Makowski (35) recently demonstrated that

the P pilus rod is a thread-like PapA fibrillar polymer that istightly wound to generate the mature helical rod. Each PapAsubunit in the pilus makes at least two contacts that are describedas head-to-tail. The tail of the subunit is defined in part by theconserved carboxy terminus of pilus subunits that is also recog-nized by the PapD chaperone (G. Soto and S.J.H., unpublishedwork). However, PapA subunits must make additional packaginginteractions to transform the thin fibrillar conformation of thePapApolymer into the single-stranded right-handed helical struc-ture. HifA subunits also contain the conserved carboxyl-terminalchaperone binding motif and are presumably held together in ahead-to-tail fashion within each strand. In addition, the HifAsubunits in each of the two left-handed helical strands mustinteract with subunits in the opposite strand to package into adouble-start right-handed helix. In this respect, the interactivesurfaces of HifA subunits may be more similar to those of actinmonomers than to PapA or other pilus subunits.Our results suggest that the H. influenzae pilus tip fibrillum

contains the minor subunit called HiMD. It is possible that theHifE protein, which is a putative second minor subunit and isencoded by the gene immediatedly downstream of hiJD (17,20), is also incorporated into the tip. HifE possesses a chap-erone binding motif at the carboxyl terminus and shareshomology with a number of minor pilus subunits (17, 20). InP pili and type 1 pili, the pilus tip contains the adhesivesubunit responsible for binding to the relevant host cellreceptor (4, 5). In this context, it is noteworthy that van Hamet al. (37) recently suggested that HifA represents theadhesive moiety in H. influenzae pili. These investigatorsbased their conclusion on the observation that E. coli DH5aharboring the hif gene cluster with a kanamycin cassette inthe hifD gene was capable of moderate attachment to humancells. The hifD insertion was believed to eliminate expressionof both hifD and the downstream hifE, such that only HifA,HifB, and HifC were being synthesized. Our demonstrationthatH. influenzae pili contain tip fibrillae raises the possibility thata minor subunit localized to the tip is the true adhesin. Experi-ments to address this issue are presently in progress.

In previous studies, insertional inactivation of hiJD resulted ina marked decrease in the number of pili (17, 20). An in-frame

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11918 Microbiology: St. Geme et al.

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20.1

FIG. 6. Purification of HifB-HifD complex and demonstration thatantiserum against Hifm reacts with purified pili. (A) SDS demonstrat-ing purified HifB-HifD complex. Lanes: 1, periplasm from E. coliC600/pMMB91 plus pTrc99A without IPTG induction; 2, periplasmfrom E. coli C600/pMMB91 plus pTrc99A induced with IPTG; 3,periplasm from E. coli C600/pJS202 plus pGK101 without IPTGinduction; 4, periplasm from E. coli C600/pJS202 plus pGK101induced with IPTG; 5, purified HifB-HifD complex; 6, purified HifB.(B) Western analysis performed with antiserum against HifD excisedfrom denaturing polyacrylamide gel. Lanes: 1, periplasm from E. coliC600/pMMB91 plus pTrc99A without IPTG induction; 2, periplasmfrom E. coli C600/pMMB91 plus pTrc99A induced with IPTG; 3,periplasm from E. coli C600/pJS202 plus pGK101 without IPTGinduction; 4, periplasm from E. coli C600/pJS202 plus pGK101induced with IPTG; 5, purified pili; 6, purified HifB-HifA complex.

deletion in hifD had a similar effect (20). These observationstogether with our finding that HifD is present at the pilus tipsuggest the possibility that this protein functions as an initiator ofpilus biogenesis, similar to the PapK and PapF proteins in P pilustips and the FimF protein in type 1 pilus tips (38-40). HifDcontains a signal peptide that is 54 amino acids long and containsa combination of structural features that make it unique. Moststriking is the presence ofboth a signal peptidase II cleavage motifat residues 17-20 and a more typical prokaryotic signal peptidefrom residues 32-54. It is possible that HifD undergoes multipleprocessing steps. Whether the amino-terminal processed regionhas specific functions in addition to being a signal peptide iscurrently being investigated.

Despite the structural differences between H. influenzae piliand P pili, we have demonstrated that Haemophilus pilus assem-bly shares a requirement for a periplasmic chaperone. Based onmolecular modeling, it appears that the H. influenzae chaperone,designated HifB, is a member of the PapD family of immuno-globulin-like chaperones (6). HifB contains all of the signaturefeatures of PapD-like chaperones, including a conserved hydro-phobic core and a conserved subunit binding cleft. Data gener-ated in the Pap system have suggested a general model in whichthe carboxy termini of newly translocated subunits zipper to theGl 3-strand of the chaperone (41). PapD-like chaperones mayprovide a platform for X3-strand zippering, allowing the subunitsto achieve their native-like conformations (C. H. Jones andS.J.H., unpublished work). These interactions explain how thechaperone controls pilus assembly by capping and uncappinginteractive subunit surfaces. The electron microscopic examina-tion of HifB-HifA complexes revealed the presence of rod-likesubassemblies, arguing that the complex is unstable. Whether thesurfaces on HifA that participate in the unique packaging inter-

actions remain partially exposed and contribute to the instabilityof the HifB-HifA complex is unknown.

In summary, H. influenzae pili share certain common fea-tures with other Gram-negative bacterial pili but are alsoclearly distinctive. Further investigation of the biogenesis ofthese structures should contribute to our understanding ofprotein-protein interactions and provide further insights intothe host-parasite relationship.

We thank D. Cutter and R. Roth for expert technical assistance. Thisworkwas supported by an American Heart Association Grant-In-Aid andan Infectious Diseases Society of America Young Investigator Award toJ.W.S. and by Public Health Service Grant AI29549 to S.J.H.

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